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First published online 3 March 2004
doi: 10.1242/dev.01056

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PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

Antonietta Salustri^1,*, Cecilia Garlanda^2,*, Emilio Hirsch^3,*, Marika De Acetis³, Alessia Maccagno¹, Barbara Bottazzi², Andrea Doni², Antonio Bastone², Giovanna Mantovani⁴, Paolo Beck Peccoz⁴, Giovanni Salvatori⁵, David J. Mahoney⁶, Anthony J. Day⁶, Gregorio Siracusa¹, Luigina Romani⁷ and Alberto Mantovani^2,8,

¹ Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome 00133, Italy
² Department of Immunology and Cell Biology, Mario Negri Institute for Pharmacological Research, Milan 20157, Italy
³ Department of Genetics, Biology and Biochemistry, University of Turin, Turin 10126, Italy
⁴ Institute of Endocrinology, Università degli Studi di Milano, Milan 20100, Italy
⁵ SigmaTau SpA, Pomezia, Rome 00040, Italy
⁶ MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK
⁷ Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia 05122, Italy
⁸ Centro IDET, Institute of General Pathology, Università degli Studi di Milano, Milan 20100, Italy

View larger version (103K): [in a new window]   Fig. 1. Defective cumulus expansion in Ptx3–/– mice. (A-D) Morphology of cumuli oophori during matrix deposition. Micrographs show individual preovulatory follicles 10 hours after hCG (A,B) and an enlargement of the enclosed cumuli (C,D). (E-H) Morphology of Ptx3+/– and Ptx3–/– ovulated cumuli immediately after isolation (E,F), or after 1 hour of in vitro culture (G,H). Scale Bars: 100 µm in A,B,E-H; 50 µm in C,D.

View larger version (63K): [in a new window]   Fig. 2. PTX3 is expressed by cumulus cells in the preovulatory follicle and localizes in the matrix. (A) In situ hybridization of an ovary isolated from a naturally cycling mouse. (B) Kinetics of Ptx3 mRNA expression in ovaries during hormonally induced ovulation. Each lane was loaded with 10 µg of total RNA. Ethidium bromide staining of the gel is shown in the lower panel. (C) Detection of PTX3 protein in ovulated intact cumuli oophori (COC), isolated cumulus cells (CC), denuded oocytes (O) and cumulus matrix (matrix) assessed by western blotting. (D) Immunofluorescence analysis (left panel) and phase contrast (right panel) of a cumulus oophorus. Scale bars: 100 µm in A; 50 µm in D.

View larger version (33K): [in a new window]   Fig. 3. PTX3 synthesis by in vitro cultured cumulus cells. (A) COCs (30/20 µl) were cultured for 16 hours in the absence (BM, basal medium) or presence of the indicated stimuli. (B) Cumulus cells isolated from 30 COCs were cultured for 16 hours in the presence of 60 oocytes or FSH, or both. PTX3 was assessed in the matrix and medium by western blotting.

View larger version (38K): [in a new window]   Fig. 4. Effects of PTX3 on matrix organization and cumulus expansion. (A) PTX3 is necessary for matrix assembly and cumulus expansion. COCs (20/20 µl) were cultured for 16 hours in the presence of FSH and FBS, and with 1 µg/ml recombinant PTX3 where indicated. HA evaluation was performed as indicated in Materials and methods. Cumulus expansion and HA distribution between matrix and medium were assessed for (a) Ptx3+/– cumuli, (b) Ptx3–/– cumuli and (c) Ptx3–/– cumuli cultured with recombinant PTX3. Reported data refer to one representative experiment of three performed. Scale bar: 100 µm. (B) Degradation of expanded cumulus matrix is not affected by recombinant PTX3. Degradation of the HA-rich matrix was assessed at 24 and 28 hours of culture by calculating the proportion of the total HA released in the culture medium, as described previously (D'Alessandris et al., 2001). In the upper panel the total amount of HA (pmol hexosamine)/COC at each time culture is reported. Error lines indicate the standard deviation of duplicate cultures.

View larger version (51K): [in a new window]   Fig. 5. Incorporation of II and PTX3 in the matrix. (A) Covalent linkage of II heavy chains to HA normally occurs in the cumulus matrix of Ptx3–/– mice. Western blot analysis, with anti-II, of Ptx3+/– and Ptx3–/– ovulated cumuli undigested and digested with hyaluronidase (ha'ase). A 85 kDa band (representing single II heavy chains) and additional high molecular weight bands (most likely representing dimers and clusters of II heavy chains) were detected after hyaluronidase digestion in both Ptx3+/– and Ptx3–/– cumulus matrix. (B) PTX3 is not covalently bound to HA in the cumulus matrix. The band recognized by anti-PTX3 shows the same intensity in undigested and digested Ptx3+/– cumuli. As expected, no PTX3 positivity was found in Ptx3–/– cumuli.

View larger version (80K): [in a new window]   Fig. 6. Co-localization of PTX3 and HA in ovulated cumuli. Ptx3+/– cumuli were stained for HA (red), PTX3 (green) and chromatin (blue), as reported in Materials and methods, and the innermost (A-D) and outermost (E-H) layers of a cumulus were photographed using a fluorescence microscope. HA histochemical staining (A,E); PTX3 immunostaining (B,F); merging of the two signals (C,G); and staining of cumulus cell nuclei (D,H). Cumuli incubated with secondary probes only showed no specific staining (data not shown).

View larger version (16K): [in a new window]   Fig. 7. Binding of PTX3 to TNFAIP6. (A) Binding of bHA (12.5 ng/well) or bPTX3 (22.2 pmol/well), or PBS as a control, to immobilized TNFAIP6 (25 pmol/well; n=3). (B) Dose-dependent binding of different amounts of soluble bPTX3 to immobilized Link_TNFAIP6 (25 pmol/well; n=4). (C) Effect of different amounts of competitors (PTX3, HA, Link_TNFAIP6) on the interaction of bPTX3 (200 ng/well) with Link_TNFAIP6-coated plates (n=4).

View larger version (17K): [in a new window]   Fig. 8. Binding of soluble PTX3 (2.2x10–6 M) to spermatozoa as assessed by FACS (A) and imunofluorescence (B). (C) Differential localization and binding of spermatozoa cultured on PTX3- or BSA-coated plastic (P=0.013; Student's t-test).

View larger version (20K): [in a new window]   Fig. 9. Expression of PTX3 in human ovarian tissues. (A) PTX3 mRNA expression by human cumulus cells in four different patients undergoing IVF. Each lane was loaded with 10 µg of total RNA. Ethidium bromide staining of the gel is shown in the lower panel. (B) Detection of PTX3 in human cumulus matrix by western blot analysis. Lane 1, human cumuli; lane 2, recombinant PTX3. (C) PTX3 protein levels in the follicular fluid of five different patients undergoing IVF. The dotted line indicates PTX3 plasma levels in healthy donors.

View larger version (15K): [in a new window]   Fig. 10. Schematic model of complex formed between PTX3 and TNFAIP6 in the cumulus matrix that could act as a node to cross-link multiple HA chains.